► Enterococcus faecalis, a gram-positive member of the mammalian gastrointestinal flora, emerged as an important contributor to nosocomial infections and antibiotic resistance gene transfer. Lipoteichoic acid…
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▼ Enterococcus faecalis, a gram-positive member of the
mammalian gastrointestinal flora, emerged as an important
contributor to nosocomial infections and antibiotic resistance gene
transfer. Lipoteichoic acid (LTA), a vital component of
gram-positive cell walls, has been reported to function in numerous
cellular processes, ranging from maintenance of cation homeostasis
and virulence to modulating function and presentation of wall
proteins such as adhesins and autolysins. Interestingly, LTA can be
covalently modified by the addition of D-alanyl ester residues,
which appear to help regulate its function by altering surface
charge. In E. faecalis the process of esterification is catalyzed
by four proteins encoded by the dlt operon. Mutants lacking a
functional dlt operon display the inability to incorporate D-alanyl
residues on LTA and are thus deficient in their ability to regulate
the anionic charge of the outer envelope in response to
extracellular cues. Recent evidence suggests that two-component
systems are responsible for sensing environmental conditions and
regulating dlt operon expression. Utilizing a reporter construct
with the upstream promoter region of dlt fused to lacZ, we were
able to determine how extracellular stimuli affect transcription of
this operon by measuring [Beta]-galactosidase activity.
Furthermore, we were able to identify specific response regulators
important for bile salt, magnesium and polymyxin B
signaling.
Advisors/Committee Members: Helmut Hirt.

Allen, D. (2008). Transcriptional
regulation of the dlt operon in Enterococcus faecalis and further
characterization of a dlta mutant. (Masters Thesis). Kansas State University. Retrieved from http://hdl.handle.net/2097/783

Chicago Manual of Style (16th Edition):

Allen, Darin. “Transcriptional
regulation of the dlt operon in Enterococcus faecalis and further
characterization of a dlta mutant.” 2008. Masters Thesis, Kansas State University. Accessed September 15, 2019.
http://hdl.handle.net/2097/783.

MLA Handbook (7th Edition):

Allen, Darin. “Transcriptional
regulation of the dlt operon in Enterococcus faecalis and further
characterization of a dlta mutant.” 2008. Web. 15 Sep 2019.

Allen D. Transcriptional
regulation of the dlt operon in Enterococcus faecalis and further
characterization of a dlta mutant. [Masters Thesis]. Kansas State University; 2008. Available from: http://hdl.handle.net/2097/783

▼ Terrapene ornata and T. carolina are closely related box turtles that live in different habitats: grasslands and desert edges, and forested areas, respectively. Considering these species' habitat selection, I predicted that T. ornata would select for higher body temperatures (Tb) and would be a more precise thermoregulator than T. carolina. I recorded time series of cloacal T b's in thigmothermal linear gradients from acclimatised (LD 12:12; 10, 20° C) box turtles. I used three analytical methods to evaluate and characterise turtles' activity: a ratio-dependent index that measured activity as an indirect function of Tb changes, a comparison of hourly mean variance of Tb (ratio-independent), and autocorrelation. I tested the thermoregulatory differences of active T. carolina and T. ornata with a factorial ANOVA, and characterised the turtles' thermoregulatory cycles with correlograms. Overall, T. ornata had significantly higher mean Tb's than T. carolina. Both species had similar diel thermoregulatory cycles with a period of approximately 24-hr. No clear differences in absolute thermoregulatory precision of Tb's were detected. These species' thermal behaviours were consistent with those reported from field studies, suggesting that there are intrinsically determined differences in thermal preferences that may help explain the different habitat choices.
Advisors/Committee Members: Hutchison, Victor H., (advisor).

► A community of ten sympatric lizard species was studied in a mountain canyon in southern Arizona to determine the effect of phylogeny on habitat use…
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▼ A community of ten sympatric lizard species was studied in a mountain canyon in southern Arizona to determine the effect of phylogeny on habitat use and thermoregulation. Habitat use, body temperatures ( Tb) and operative environmental temperatures ( Te) were recorded in the field, as well as the selected temperature range (Tsel) for active lizards in the laboratory. Te varied significantly with elevation, time of day, macrohabitat, and microhabitat with most of the variation occurring at the microhabitat level, indicating a small spatial scale of thermal heterogeneity at the study site. At most times of day and in most macrohabitats, Te bracketed Tsel for the species indicating that maintenance of Tb within Tsel was possible during most daylight hours. Randomization analyses of matrices of phylogenetic similarity, habitat use and microsite temperature overlaps showed significant correlations between phylogenetic similarity, habitat use, and field active Tbs. There was no correlation between phylogenetic similarity and T sel. Indices of thermoregulation calculated for each species revealed that although these lizards thermoregulated effectively, there was no correlation between the matrix of phylogenetic similarity and similarity matrices for accuracy or effectiveness of thermoregulation. Pseudocommunity analyses showed no partitioning of thermal resources, indeed species tended to use similar thermal resources despite disparate habitat use. The significant effect of phylogeny on microhabitat use, and field Tb but not on Tsel suggests that microhabitat selection may override thermoregulation, possibly due to tradeoffs between thermoregulation, foraging, predator avoidance or reproduction.
Advisors/Committee Members: Vitt, Laurie J., (advisor).

► Animals that received the high dose of pyrogen + antipyrogen at midnight had higher thermoregulatory precision than those injected at noon on day 1. Control…
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▼ Animals that received the high dose of pyrogen + antipyrogen at midnight had higher thermoregulatory precision than those injected at noon on day 1. Control animals had higher precision than those injected with the high dose of pyrogen + antipyrogen and those injected with the high dose of pyrogen only on day 1. Midnight injections produced longer responses than did noon injections, and higher doses induced faster responses.
Advisors/Committee Members: Hutchison, Victor H., (advisor), Gaffin, Douglas, (advisor).

Deen, C. M. (2006). Effects of lipopolysaccharide, antipyrogen, and body condition on the chronopharmacology of fever in Dipsosaurus dorsalis. (Doctoral Dissertation). University of Oklahoma. Retrieved from http://hdl.handle.net/11244/1043

Chicago Manual of Style (16th Edition):

Deen, Cari Melissa. “Effects of lipopolysaccharide, antipyrogen, and body condition on the chronopharmacology of fever in Dipsosaurus dorsalis.” 2006. Doctoral Dissertation, University of Oklahoma. Accessed September 15, 2019.
http://hdl.handle.net/11244/1043.

Deen CM. Effects of lipopolysaccharide, antipyrogen, and body condition on the chronopharmacology of fever in Dipsosaurus dorsalis. [Doctoral Dissertation]. University of Oklahoma; 2006. Available from: http://hdl.handle.net/11244/1043

►Osmoregulation is facilitated by using an assortment of ion and water channels to assist acclimation to changing conditions and to maintain cellular homeostasis. Euryhaline…
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▼Osmoregulation is facilitated by using an assortment of ion and water channels to assist acclimation to changing conditions and to maintain cellular homeostasis. Euryhaline fish can be found in both seawater and freshwater (SW and FW) environments. Expression of ion and water channels have been experimentally demonstrated to change as a fish acclimates to different environmental conditions. Relatively recently, a new group of water channels has been discovered that are primarily intracellular and includes aquaporin 11 (AQP11). Typically, AQPs are located on the cell plasma membrane to allow water to flow in and out of the cell by osmosis to assist in cellular homeostasis. Since AQP11 has been primarily located in the membrane of the endoplasmic reticulum, it was expected that the expression of AQP11 would not be affected by the acclimation of fish to SW. For this study, Anguilla rostrata (the American eel) was selected partly because it has two paralogs of AQP11 (AQP11a and AQP11b). Eels were subjected to a SW-acclimation experiment where they were acclimated to SW for predetermined times, while concurrent FW control samples were also kept. Initially, genomic sequences for AQP11a and AQP11b genes were available for the closely related Anguilla japonica (the Japanese eel), thus cloning and sequencing of the AQP11a and 11b paralog cDNA sequences from the American eel were performed. The obtained sequences were used to generate primers and antibodies for further studies. Quantification of mRNA/cDNA was performed using quantitative PCR (qPCR). The analysis of the qPCR data determined that both AQP11a and AQP11b had significant differences between FW and SW in the expression of mRNA/cDNA in multiple osmoregulatory tissues (i.e. kidney, gill, and gastrointestinal tract). Quantification of AQP11 proteins was facilitated using western blotting. The antibody generated for AQP11a was found to be non-specific and unusable for quantification potentially due to a possible PDZ domain located at the C-terminal end where polypeptides for the antibody were made. The antibody made against AQP11b produced quantifiable western blots. Significant differences between AQP11b bands were found in all intestinal tissues. Additionally, localization of AQP11 paralogs was carried out using immunohistochemistry in multiple intestinal tissues as well as kidney and liver in both FW and SW-acclimated fish. In SW tissues AQP11a and AQP11b were seen to be present in smooth muscle in intestinal tissues as well as on the luminal side of epithelial cells and expression of AQP11a and AQP11b seems to be lower in corresponding FW tissues. In kidney tissue AQP11b was occasionally present in nephron tubules in SW tissue but not FW tissue, and AQP11b was also found in the liver surrounding the liver ducts. The within-group and between-group quantitative analysis of the FW and SW-acclimated AQP11 paralogs transcript and protein expression suggests that AQP11 paralogs are affected by SW-acclimation even though they are intracellular. These results may also…
Advisors/Committee Members: Johanne Lewis, Vinoth Sittaramane.

Managers require effective methods for forecasting the impacts of introduced species, so that invasion threats can be prioritized. An emerging tool for invasive species risk…
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Managers require effective methods for forecasting the impacts of introduced species, so that invasion threats can be prioritized. An emerging tool for invasive species risk assessment is the systematic comparison of the functional response (the relationship between predation rate and prey density) of native and invasive species. This approach is based on the assumption that species that have higher resource consumption rates will be more disruptive to food webs. Here, I use functional response analysis to examine the influence of biotic interactions on per capita effects of two congeneric North American crayfishes (Orconectes limosus and O. virilis) that have extensive invasion histories throughout this continent and in Europe. By experimental testing geographically disparate populations, I found intraspecific variation in the functional response curves and maximum feeding rates. A second set of experiments compared O. limosus and O. virilis from their respective native and invaded ranges, and from populations that are sympatric and allopatric with one another; these experiments further revealed interspecific and intraspecific variation in functional responses. Finally, I tested the effects of perceived competitors on the functional responses of both crayfishes. The presence of conspecific and heterospecific crayfish suppressed the maximum feeding rate of O. limosus, but they had no effect on the feeding rate of O. virilis. I conclude that source population and interspecific interactions mediate per capita effects, and possibly the overall impact, of introduced crayfishes. My results caution against the use of single populations in conducting invasive species risk assessments.

Microtubules are long polymers that form an integral part of the cytoskeleton, an intracellular, dynamic scaffold responsible for coordinating cell movement, intracellular transport, and cell…
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Microtubules are long polymers that form an integral part of the cytoskeleton, an intracellular, dynamic scaffold responsible for coordinating cell movement, intracellular transport, and cell division. Misregulation of the microtubule cytoskeleton can lead to various developmental problems and has been implicated in certain cancers. Microtubules can be studied in vitro and such experiments have been highly informative as to the molecular basis of their growth dynamics. To gain a deeper understanding, however, microtubules must be studied in vivo, in the context of the complete cell. In contrast to in vitro experiments, there is no simple and straightforward way to analyze the data that result from in vivo experiments. Here, using readily-available software, I have built up a microtubule tracking protocol that goes from raw data (time-lapse videos of growing microtubules) to statistical summaries of microtubule growth rates. The protocol is flexible, straightforward, time-efficient, and mostly automatic.

► Cellular morphology depends on a complex network of microtubules, which are long, hollow polymers made up of a/b-tubulin subunits. Microtubules are not fixed structures; rather,…
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▼ Cellular morphology depends on a complex network of microtubules, which are long, hollow polymers made up of a/b-tubulin subunits. Microtubules are not fixed structures; rather, they are highly dynamic, constantly being built, taken apart, and changing in shape and location to suit the needs of the cell. This thesis aims to answer some of these questions. Microtubule growth is accelerated by enzymes such as XMAP215, but in vivo microtubule growth rates remain much higher than in vitro reconstitution assays using purified components. Recently, XMAP215 and EB1 have been shown to synergistically enhance microtubule growth to near physiological rates. The growth rates reported remain lower, however, than those observed in C. elegans embryos and the theoretical upper limit derived from mass-transfer models. We sought to determine the effects of macromolecular crowding agents on microtubule growth rates. We found that the apparent rate constant for tubulin addition increased up to 10-fold inviscous environments with large macromolecules. In contrast, increasing the viscosity with small solutes decreased growth rates in a manner consistent with tubulin binding to microtubule ends in a diffusion-limited reaction. Adding crowding agents with XMAP215 and EB1 resulted in growth rates that saturated at 45 um min−1 at 10 uM tubulin. To our knowledge, this represents the fastest in vitro microtubule growth rates measured to date and approaches the theoretical limit. Microtubules are born and reborn continuously, even during quiescence, but nucleation is arguably the least understood aspect of microtubule dynamics. Using single-molecule biophysics, we show that nucleation from g-TuRCs, axonemes, and seed microtubules requires tubulin concentrations that lie well above the critical concentration yet do not support spontaneous nucleation. We measured considerable time lags between the arrival of tubulin and the onset of steady-state elongation. Microtubule associated proteins (MAPs) alter these time-lags. Catastrophe factors (MCAK and EB1) inhibited nucleation, while a polymerase (XMAP215) and an anti-catastrophe factor, which we have characterized for the first time (TPX2), both promoted nucleation. We observed similar phenomena in cells: depleting soluble tubulin levels in cells with tubulin-sequestering drugs reduced centrosomal nucleation rates. We conclude that GTP hydrolysis inhibits microtubule nucleation by destabilizing the nascent plus ends required for persistent elongation. Given its importance for the survival of cells, it is not surprising that tubulin is an important pharmacological target. Several drugs used to treat cancer patients directly target microtubules. Some drugs stabilize microtubules, preventing disassembly, while others destabilize microtubules, but the resulting mechanism of action is generally the same: a block in M phase progression and subsequent cell death. The use of effective dosages of these drugs is often limited, however, by severely toxic side effects on non-cancerous cells. Diazonamide A (DZA)…
Advisors/Committee Members: Gary Brouhard (Supervisor).

The regulation and management of lake water levels have become a major form of anthropogenic disturbance in temperate and boreal aquatic ecosystems. Drawing the water…
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The regulation and management of lake water levels have become a major form of anthropogenic disturbance in temperate and boreal aquatic ecosystems. Drawing the water level down as the winter season progresses can lead to substantial exposure of the littoral zone, rendering it susceptible to desiccation and freezing. Such changes in the hydrological regime of littoral zones can have significant effects on aquatic biota, including macroinvertebrates. These organisms are widely considered as robust bioindicators, but it is also well known that their abundance and distribution is characterized by substantial patchiness. Often, scientists who use macroinvertebrates as bioindicators, focus their sampling on a single habitat type (e.g., stony littoral) to reduce the heterogeneity in macroinvertebrate distributions. However, applying such an approach in the context of an environmental stressor makes it difficult to draw general conclusions about the impact of a potential stressor on the entire littoral zone. Recently, a debate has emerged in the literature over the effects of water level drawdown on macroinvertebrate communities and we think that at least part of this debate may be due to different sampling strategies employed across studies to examine the effects of drawdown on macroinvertebrate communities. In order to evaluate how nearshore macroinvertebrate communities, from a variety of habitats, are affected by drawdown, we sampled 15 reservoirs and quantified macroinvertebrate abundance and composition. To these data, we applied a combination of (generalized) linear mixed effects models and multivariate analyses to answer the following question; what are the effects of drawdown on macroinvertebrate abundance and community composition? To address this question and incorporate the possible effects of heterogeneity in abundance and distribution of macroinvertebrates, we explicitly considered multiple physical environmental variables as additional predictors of macroinvertebrate abundance and community composition. We found that drawdown and thermal regime were significant predictors of macroinvertebrate abundance, but explained negligible amounts of variation in the community composition. Overall, these findings help elucidate how macroinvertebrate communities respond to winter drawdown and strengthen the knowledge on how localized environmental conditions influence macroinvertebrates. Furthermore we think that our sampling and analytical approach provides a useful template for other scientists to adopt when quantifying the effect of a potential stressor on nearshore macroinvertebrate communities.

Declines in amphibian populations have been noted since the late 1980's with many of the causes linked to habitat contamination and destruction by natural and…
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Declines in amphibian populations have been noted since the late 1980's with many of the causes linked to habitat contamination and destruction by natural and anthropogenic sources. Amphibians with bi-phasic life histories have been thought to be particularly vulnerable to negative environmental conditions but there is much evidence of their resilience and capacity to survive in degraded or contaminated environments. Larval amphibians in small permanent or ephemeral ponds may be particularly vulnerable to altered environmental conditions and may face the greatest pressure to adapt. In Chapter 1, I briefly review the evidence of amphibian declines and how populations may adapt to and overcome assorted negative environmental factors. In Chapter 2, I investigated the phenomenon of adaptation in a particular circumstance. I collected Spotted Salamander (Ambystoma maculatum) egg masses from a population breeding in a naturally acidic pond called Bat Lake, as well as four other populations living in lakes closer to pH neutral. To determine if the salamanders were adapted to their particular breeding lakes, I used common garden experiments in the lab where I raised larvae from each of the lakes in the waters from all the other lakes, as well as their own and compared the survival rates, size at metamorphosis and time to metamorphosis. Bat Lake larvae grew larger and survived longer in their acidic native breeding pond water than in the waters of other lakes suggesting they are adapted to their native pond waters. The larvae from the other lake populations showed similar results and fared better in their native waters than in the Bat Lake water. Based on these results, in Chapter 3, I tested if pH, specifically, was causal. I raised larvae from the five populations in waters of different pH (4.0, 5.5, 7.0), reasoning that if the Bat Lake population was more adapted to the high acidity compared to the other populations, the larvae from Bat Lake should demonstrate higher fitness correlates at lower pH than larvae of other populations. In this experiment, the Bat Lake larvae survived longer and grew larger in the highly acidic water than did larvae from the other populations. My results are consistent with natural selection for tolerance of low pH conditions tolerance among Bat Lake Spotted Salamander larvae and indicate that the salamanders from Bat Lake potentially exhibit signs of local adaption to the highly acidic conditions in which they live. This study was unable, however to prove whether this adaptation was due to evolutionary change, phenotypic plasticity, maternal effects or some other unknown factor.

The stereotyped meiotic pairing and the subsequent synapsis that keeps the homologous chromosomes together for the shuffling of genetic material to take place is central…
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The stereotyped meiotic pairing and the subsequent synapsis that keeps the homologous chromosomes together for the shuffling of genetic material to take place is central to meiosis. In the nematode Caenorhabditis elegans, HTP-3 holds a vital role in the assembly of the synaptonemal complex (SC) that polymerizes throughout the length of the homologs during synapsis. HTP-3 has established roles in pairing and synapsis, but also in double strand break (DSB) formation, a necessary first step in the aforementioned exchange of genetic material. HTP-3 contains a HORMA domain, which is a protein-protein interaction domain conserved amongst proteins with roles ranging from apoptosis to the spindle assembly checkpoint (Aravind, 1998; Jao, 2013). We know that HTP-3 has a central role in meiosis, but the molecular mechanism by which it performs its functions is unclear because the protein is involved in so many processes. Characterization of the htp-3(vc79) allele, which contains a mutation in the conserved HORMA domain of HTP-3, has allowed me to dissect the functions specifically of the HORMA domain regarding the numerous roles of HTP-3. HTP-3 loading on the SC is abrogated in the htp-3(vc79) mutant, where instead meiotic aggregates of HTP-3 can be observed in pachytene nuclei. Analysis of this mutant uncovered that an intact HORMA domain in HTP-3 is necessary for its co-loading on the axis with REC-8, a component of the cohesion complexes that keep the sister chromatids together until meiosis II. However, DSBs still occur in the mutant indicating that its HORMA domain is dispensable for this particular function. Further experiments could investigate how the mutation in htp-3(vc79) affects the binding partner landscape of HTP-3 in order to shine light on meiotic processes such as pairing, synapsis and DSB formation.

Prions are abnormally folded proteins that are able to self-propagate and transmit its conformation to other copies of the same protein. They are usually comprised…
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Prions are abnormally folded proteins that are able to self-propagate and transmit its conformation to other copies of the same protein. They are usually comprised of fibrillar, β-sheet-rich structures called amyloid, which forms insoluble aggregates and impairs the normal function of the protein. Prions in Saccharomyces cerevisiae can propagate heritable phenotypes, function in large-scale gene regulation, and uncover hidden genetic variation. They have also suggested conversion mechanisms that are applicable to mammalian and human prion diseases. Almost all of the prions have an asparagine/glutamine-rich (N/Q-rich) composition. In addition, there are around 200 other S. cerevisiae proteins with prion-like N/Q-rich sequence composition. In general, these prion-like proteins can be divided into those that are N-rich and Q-rich, with the former being better prion formers experimentally. In this thesis project, the emergence and evolution of these yeast prion-like proteins is investigated. N/Q-rich proteins have come to a larger prominence since the last common ancestor of Saccharomycetes, especially N-rich ones. Mutational bias leading to formation of poly-N tracts may have triggered the prion phenomenon early in the evolution of Saccharomycetes. In contrast, some clades, such as Eurotiales, have lost most of its N/Q-rich proteins, possibly due to some mechanistic intolerance to the aggregation and propagation of amyloids. Additionally, there are two species within the Saccharomycetes clade, Ogataea parapolymorpha and Ashbya gossypii, with very low numbers of N/Q-rich proteins. The low percentages of guanidine and cytidine may have provided a reason for this, with a combination of mutational and deleterious effects.

Freshwater resources are experiencing many broad-scale anthropogenic pressures that overlap in space and time. Hydrological modifications from damming and subsequent reservoir operation, such as winter…
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Freshwater resources are experiencing many broad-scale anthropogenic pressures that overlap in space and time. Hydrological modifications from damming and subsequent reservoir operation, such as winter water-level drawdowns, have become a major anthropogenic disturbance across temperate and boreal aquatic ecosystems. Although drawdown has widely been recognized to impair littoral and shallow ecosystem structure and functioning, there has been little investigation of the long-term implications on water quality in deep aquatic systems. I used a paleolimnological "snapshot" technique (i.e. a top-bottom design) to investigate the pre-dam to contemporary trends of water quality across 12 temperate reservoirs experiencing annual winter drawdown. In addition, I conducted detailed analyses of sediment cores from Grand Lac Saint-François (Québec, Canada) to draw further insight into how changes in hydrology could alter the dynamics of reservoir water quality. Based on correlative and multivariate analyses, I did not find strong evidence for drawdown as the primary driver of water quality change in our regional survey. Instead, reservoir morphometry and watershed characteristics (i.e. geography, maximum depth, and cropland areas) appeared to be stronger drivers of long-term water quality trends than drawdown amplitudes. Based on the higher resolution analyses of biological proxies at Grand Lac Saint-François (i.e. sedimentary pigments and diatom metrics), I detected significant trends over the last century, which is indicative of declining water quality conditions. These trajectories in water quality, as well as an observed increase in variability, coincided with dam construction and the onset of water level drawdown. However, due to the high ecological variability and the limited time frame of monitoring data, I was unable to conclusively associate drawdown levels with the declining water quality trends at Grand Lac Saint-François. In contrast, watershed nutrient surpluses and warming temperatures were identified as significant explanatory variables of the water quality metrics. This work highlights the need for long-term and/or high-resolution data in order to better interpret ecological change amongst multiple stressors. Water quality management can be complicated due to the diverse functions and operational strategies across reservoirs, as well as the persistence of increasing anthropogenic stressors. As such, paleolimnology can be a powerful tool in addressing the many ecological concerns in contemporary reservoir management. Understanding water quality dynamics in reservoirs with different functions, and identifying how these operations may exacerbate (or mitigate) declining water quality conditions is crucial to safeguarding freshwater resources in this era of multiple stressors.

Loss of retinoblastoma protein (pRB) function is one of the most common steps in cancer pathogenesis in humans. pRB regulates E2F-family transcription factors required for…
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Loss of retinoblastoma protein (pRB) function is one of the most common steps in cancer pathogenesis in humans. pRB regulates E2F-family transcription factors required for expression of genes that drive cell cycle progression. Consequently, understanding and developing treatments to reduce unregulated cellular proliferation in pRB-related cancers relies largely on identifying and exploiting factors that cooperate with RB mutations. The EGFR-regulated transcriptional repressor Capicua is one such factor which cooperates with loss of the pRB homologue Retinoblastoma-Related Factor-1 (RBF1) in Drosophila melanogaster. Loss-of-function Capicua mutations in the eye imaginal disc promote ectopic proliferation and survival of Rbf1 mutant cells specifically. For this reason identifying genes directly targeted by Capicua de-repressed in the context of an Rbf1 mutant background may identify novel factors responsible for survival and proliferation of cancer cells. Despite well characterized transcriptional repression during embryogenesis and imaginal disc development, there is little evidence of directly bound Capicua target genes in Drosophila. RNA-sequencing data obtained from eye imaginal discs was used to identify possible direct targets of Capicua. These candidate genes were identified by analysis of mRNAs commonly upregulated in Capicua mutant discs, in both a wild-type and Rbf1 mutant background. Investigation of Capicua's DNA-binding activity using Chromatin Immunoprecipitation (ChIP) assays, the gene Phosphoribosyl Pyrophosphate Synthetase (PRPS) was identified as a direct target. Direct binding evidence by ChIP was achieved by the generation and Gal4 driven expression of a Capicua cDNA construct. Overexpression of Capicua also provided additional insights into the general effects this transcription factor has on cell cycle progression. Investigation of Capicua's recruitment to polytene chromosomes concluded that constitutive expression throughout development is necessary to exhibit stable DNA-binding activity. When this requirement is met salivary gland and polytene chromosome size is reduced, consistent with interruption of endocycle progression. This supports prior studies citing the role of Capicua in cellular proliferation and is consistent with direct binding and likely repression of the nucleotide biosynthetic gene PRPS. Furthermore, identification of a direct interaction with PRPS suggests this factor is implicated in the survival and proliferation of RBF1 deficient cells.

Spontaneous mutations are the ultimate source of genetic variation, which can generate phenotypic variation upon which natural selection can act. Understanding the rates, patterns, and…
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Spontaneous mutations are the ultimate source of genetic variation, which can generate phenotypic variation upon which natural selection can act. Understanding the rates, patterns, and fitness effects of mutation is essential to many fields of biology, thus several studies have attempted to investigate this fundamental phenomenon over the years. However, knowledge is still limited regarding the mutation rates in most organisms as well as the way selection acts on new mutations in a population. My thesis seeks to increase the understanding of the evolutionary phenomena of mutation and selection by analysing the genomes of mutation accumulation (MA) lines of Daphnia pulex maintained under selection-minimized conditions for many generations as well as isolates from a laboratory population that was founded with the same asexual progenitor and was maintained with selection acting throughout the course of the experiment. This unique experimental setup allows comparison of the rates, types, and patterns of mutations accumulated in conditions with and without selection. The Daphnia were propagated asexually, which allowed the detection of new mutations in a heterozygous state as well as large-scale mutations that result in loss of heterozygosity (LOH). Whole genome sequencing of 24 MA lines facilitated the detection of 477 single nucleotide mutations, and I found that the overall mutation rate in Daphnia is similar to that of other metazoans. One MA line experienced a massive LOH event that caused complete homozygosity across an entire chromosome (3% of the genome), resulting from a large gene conversion event. I also sequenced six isolates from the laboratory population and found fewer mutations than expected, demonstrating that purifying selection was acting strongly in order to purge harmful mutations that decrease fitness. Surprisingly though, the population maintained a high level of genetic diversity, with four distinct lineages from only six individuals. This observed pattern of high diversity was likely driven by balancing selection. My work challenges the assumption that selection is inefficient in asexual populations, provides an example of high diversity maintenance, and provides insight into the entire spectrum and implications of mutation in Daphnia.

Meiotic recombination initiates with the introduction of programmed double-stranded breaks (DSBs) in most species. Only a limited number of DSBs are processed into crossovers (COs).…
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Meiotic recombination initiates with the introduction of programmed double-stranded breaks (DSBs) in most species. Only a limited number of DSBs are processed into crossovers (COs). In Caenorhabditis elegans (C. elegans), each pair of homologous chromosomes receives only one CO due to CO interference. These obligate COs then manifest as cytologically visible physical attachments called chiasmata, which ensure proper homolog segregation during meiosis I. Previous analyses have revealed that C. elegans ZHP-3 is an ortholog of S. cerevisiae Zip3p and is required for crossover formation (Jantsch et al. 2004). We have identified three additional ZHP-3 (three) paralogs: ZTP-1, ZTP-2 and ZTP-3, all of which contain a type of RING finger usually found in E3 Ubiquitin or SUMO ligases. We have discovered that ZTP-2, like ZHP-3, localizes to the synaptonemal complex (SC) at early pachytene and is then restricted to presumptive CO sites at late pachytene. ZTP-2 and ZHP-3 colocalize and are codependent for their recruitment to the SC and CO sites. Given the known involvement of ZHP-3 in crossover formation, we investigated a possible role of ZTP-2 in this process by examining two ztp-2 mutants: the predicted null mutant ztp-2(vv103) and the hypomorphic mutant ztp-2(vv96). Both mutants displayed mislocalization of ZHP-3, high embryonic lethality and a high incidence of males, collectively indicative of a defect in crossover formation. While ztp-2(vv103) mutants predominantly exhibited 12 univalents at diakinesis, consistent with a severe defect in chiasma formation, ztp-2(vv96) mutants retained 30% of the wild-type level of genetic exchange, and on average exhibited 8-9 DAPI bodies at diakinesis, consistent with the interpretation that the mutant is competent for crossovers at reduced levels. Given that both mutants exhibit similar levels of embryonic lethality (96% for ztp-2(vv103) and 93% for ztp-2(vv96)), we hypothesize that the physical connections that form as a result of recombination between homologs in the hypomorph are not sufficient to promote proper segregation at metaphase I, this is further demonstrated by the presence of DSB-dependent tethered univalents at diakinesis. Consistent with this interpretation, the late stage CO marker COSA-1 (Yokoo et al. 2012) was not detectable in late pachytene in both ztp-2(vv103) and ztp-2(vv96) mutants. We propose that ZHP-3 and ZTP-2 form a heterodimeric E3 ligase complex which is responsible for CO specification and chiasma formation.

Nguyen TNH. Characterization of ztp-2, a member of a family of RING- finger domain-containing proteins required for crossover formation. [Masters Thesis]. McGill University; 2016. Available from: http://digitool.library.mcgill.ca/thesisfile143797.pdf

McGill University

17.
Yee, Callista.
Investigation of the mechanisms that control the pro-longevity response to mitochondrial reactive oxygen species.

In Caenorhabditis elegans, two mutations that affect mitochondrial electron transport chain subunits (isp-1 and nuo-6) result in increased mitochondrial reactive oxygen species (mtROS). These mutations…
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In Caenorhabditis elegans, two mutations that affect mitochondrial electron transport chain subunits (isp-1 and nuo-6) result in increased mitochondrial reactive oxygen species (mtROS). These mutations cause a significant increase in lifespan relative to the wild type. Treatment with the pro-oxidant paraquat (PQ) can also significantly increase wild type lifespan, but is not additive to the lifespan of the two mutants. Using gene arrays, we determined a large overlap of differentially expressed genes between isp-1, nuo-6 and PQ treatment. These and other results are contrary the Free Radical Theory of aging and suggest that increased levels of mtROS act as a signal to extend lifespan in C. elegans. We wanted to understand how mtROS signaling is sensed and transduced in order to elicit these changes in gene expression. Many processes require components of the intrinsic apoptotic pathway to perform tasks that do not result in apoptosis (a type of cell death), for example, aspects of cell cycle regulation. Activation of the pathway by an elevation of mtROS does not affect apoptosis but protects from the consequences of mitochondrial dysfunction by triggering a unique pattern of gene expression that modulates stress sensitivity and promotes survival. In vertebrates, mtROS induce apoptosis through the intrinsic pathway to protect from severely damaged cells. Our observations in nematodes demonstrate that sensing of mtROS by the apoptotic pathway can, independently of apoptosis, elicit protective mechanisms that keep the organism alive under stressful conditions. This results in extended longevity when mtROS generation is inappropriately elevated. These findings clarify the relationships between mitochondria, ROS, apoptosis, and aging.

Modern geological time is commonly referred to as the Anthropocene; a designation recognizing the extent to which humans dominate processes and life on Earth. Within…
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Modern geological time is commonly referred to as the Anthropocene; a designation recognizing the extent to which humans dominate processes and life on Earth. Within this context, a major theme of biodiversity research is to model and predict species losses due to land exploitation and use. However, in order to more completely understand the effect of human stressors on biodiversity, species losses and gains along with biodiversity change over varied temporal and spatial scales need to be considered in concert. My research seeks to fulfill two main objectives related to both biodiversity trends throughout the Anthropocene and the expansion of paleolimnological techniques for biodiversity science. Firstly, by paying closer attention to the way in which beta diversity can uncover trends previously missed when examining alpha or gamma diversity alone, my work helped improve our understanding of freshwater biodiversity responses to the anthropogenic stressors that have accelerated over the last ~ 150 years. Secondly, by integrating paleolimnological data with data collected from contemporary timescales, and with the application of DNA-based approaches to paleolimnology, I answered questions novel to both paleolimnology and biodiversity science. In my first chapter, I used diatom assemblage data from the U.S. Environmental Protection Agency's National Lakes Assessment (NLA) program to compare the variation in diatom assemblages across environmental and spatial gradients, using both water-column and surface sediment data. Here I showed that diatom assemblages from both types of sampling were characterized by environmental and spatial gradients in similar ways. In my second chapter, I extended this work with the NLA data, examining modern and historical (pre-1850 CE) timeframes, and showed that beta diversity responded strongly to national-scale land use gradients, with turnover hotspots in regions with low forest cover. My third chapter focused on a specific stressor for aquatic biodiversity, metal contamination in an iron-ore mining region of northern Québec, and showed that the beta diversity of zooplankton communities responded strongly to heavy metal loading. Finally, in my fourth chapter I used metabarcoding approaches to more fully characterize microbial eukaryote communities from sediment cores in this same mining region and showed substantial temporal beta diversity in both diatoms and green algae. This final chapter was the capstone for this work, continuing the integration of paleolimnological data with DNA-based approaches, capturing a more complete representation of aquatic biodiversity than possible with individual proxies. I also demonstrated how beta diversity is an important way to characterize diversity in systems experiencing multiple stressors. In general, this research provides insight into the importance of multi-scale and multi-metric methods in the study of aquatic biodiversity, while illuminating key drivers of aquatic assemblages through time.

Human-mediated biological invasions are one of the main threats to freshwater systems, contributing to the escalating biodiversity loss in these habitats. Some of the most…
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Human-mediated biological invasions are one of the main threats to freshwater systems, contributing to the escalating biodiversity loss in these habitats. Some of the most dramatic and well-studied ecological impacts in aquatic communities have been linked to the introduction of predators and their effects on native prey communities. However, invasive predators are also likely to have strong effects on native predators, through competition and/or intraguild predation, resulting in ecological changes such as dietary and trophic shifts in the native species. In this study, I focused on the predatory peacock bass (Cichla monoculus, Family Cichlidae) that was introduced into Lake Gatun, Panama in the late 1960's, and explored its potential effects on the diet and trophic ecology of a native predatory fish, Hoplias microlepis (Family Erythrinidae). Specifically, I tested the hypothesis that H. microlepis diversified its diet and broadened its niche in the presence of the peacock bass in Lake Gatun. To do so, I first quantified the dietary niche of both the introduced and native predators in sympatry. Second, I compared the dietary niche of the native predator in the presence vs. absence of the introduced peacock bass. I used a combination of stomach content analysis and stable isotope analysis to estimate the dietary niches. I found evidence to suggest that in the presence of the peacock bass, H. microlepis diversified its diet and expanded its isotopic niche. The isotopic niches of H. microlepis and C. monoculus were generally similar in size and overlapped significantly across sites in Lake Gatun, except for one site (Chagres) where overlap was moderate. An isotopic niche overlap between species suggests that they may be feeding on similar resources, however H. microlepis included more invertebrate prey in its diet than C. monoculus. In addition, scavenged fish chunks comprised 26% of the diet of H. microlepis in Lake Gatun, but scavenging was not recorded in H. microlepis in the absence of C. monoculus nor in C. monoculus. Scavenged fish chunks consisted of peacock bass and Oreochromis niloticus (Nile tilapia) remains, both heavily harvested, non-native species in Lake Gatun. In the three main sites that we sampled in Lake Gatun, it is common for fishers to return scraps of fish filleted at the docks into the lake where H. microlepis presumably scavenges on them. Although the extent of this pattern across Lake Gatun as a whole is currently unknown, I propose that at given locations the invasive peacock bass indirectly facilitates the native predator by providing a trophic subsidy and that this is human-mediated through fishing. This study highlights the complexity of interactions occurring in invaded systems, as well as the importance of using complementary approaches and incorporating the human dimension in their study.

The oocyte reserve is finite because all oocyte precursors stop proliferation and enter meiosis in early fetal life. In addition, 70% of the initial oocyte…
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The oocyte reserve is finite because all oocyte precursors stop proliferation and enter meiosis in early fetal life. In addition, 70% of the initial oocyte population is eliminated perinatally. During this period, the oocyte goes through Meiotic Prophase I (MPI), wherein homologous chromosomes synapse and recombine. Since an error in meiotic synapsis would result in aneuploidy, it has been hypothesized that oocytes with meiotic errors are eliminated by a surveillance mechanism. Previous studies have used the XO and the XY sex-reversed female mice, in which the single X chromosome is missing its pairing partner, to test this hypothesis, but did not reliably quantify the extent of oocyte loss in these models after birth when the oocyte reserve establishes. Using two novel immunofluorescence-based methods to identify and count oocytes in both dissociated and wholemount ovaries, my results show that XO and XY ovaries retained 15% the number of oocytes as their XX control ovaries did four days after birth. Co-staining with anti-gammaH2AFX (phosphorylated histone H2AFX, an indicator of DNA damage or chromosome asynapsis) revealed that XO and XY ovaries possessed thrice as many oocytes carrying unsynapsed chromatin on the day of birth compared to their XX counterparts. These gammaH2AFX-positive oocyte populations greatly diminished, concurrent with the excessive oocyte loss during four days after birth. Furthermore, one day after birth, XO and XY ovaries possessed three times more oocytes positive for cleaved-PARP1, an indicator of apoptosis. Taken together, our results suggest that oocytes with synaptic errors marked with gammaH2AFX are eliminated by apoptosis.

► Global average temperature is rapidly increasing and is currently the warmest it has been in the Northern Hemisphere over the past centuries, driving species to…
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▼ Global average temperature is rapidly increasing and is currently the warmest it has been in the Northern Hemisphere over the past centuries, driving species to local extirpation or extinction, to shift their distributional range or to adapt locally to the warming climate. A given species' sensitivity to changes in its environment may be related with patterns and amount of variation in its phenotype. As such, a decrease in body size has been proposed as the third universal ecological response to global warming, following distributional shifts and changes in phenology. Across their geographic ranges, mammals are thought to vary in body size following Bergmann's rule, which predicts that populations within a species found at higher latitudes, or colder climates, are larger in size compared to populations occurring farther south in warmer temperatures. While Bergmann's rule is well supported by empirical data among mammals, there is more and more evidence for exceptions to the rule, with species either decreasing in size with latitude or displaying no apparent relation between latitude and size. The patterns of variation in various morphological traits (skull length, skull width, tooth row length, and body mass) of 17 small and midsize mammalian hosts of Lyme disease were analyzed to determine if body size variation occurred in a predictable manner through space and time at an interspecific and intraspecific level. Results suggest little evidence of a generalizable pattern supporting Bergmann's rule within and among species at both a broad spatial and temporal scale. The effect of latitude or time on each of the morphological traits studied were highly variable leading to three types of responses: increases in size, decreases in size, or no changes in size across space and time. Overall, size trends were detected more often in space than through time, as size variation in space was studied over a significantly larger temperature gradient than the recent change in temperature that occurred over the past 120 years. Additionally, large-bodied species were not more likely to conform to Bergmann's rule than small-bodied species, in contrast to what was previously reported in the literature; in fact, this study showed that small mammals were found to vary more in size with latitude or time than midsize mammals. Contrary to predictions, size trends related to cranial measurements were detected as more likely to conform to Bergmann's rule than body mass, indicating the importance of simultaneously comparing metrics in studies on body size variation across species' ranges. However, body mass was found to have increased amounts of trait variability compared to cranial measurements; along a latitudinal gradient, the direction and magnitude of the variability depended on the size category of the species. Climate change is expected to cause the mammalian hosts of Lyme disease to expand their geographic ranges northward, facilitating the establishment of the bacteria and tick populations in southern Canada. For the mammalian hosts of…
Advisors/Committee Members: Virginie Millien (Internal/Supervisor).

Within metacommunity theory, stability of ecosystems is a fundamental concept. Synchrony between populations is known to cause greater regional variability and diminish the possibility of…
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Within metacommunity theory, stability of ecosystems is a fundamental concept. Synchrony between populations is known to cause greater regional variability and diminish the possibility of rescue effects after a catastrophe, and thus it negatively affects stability. However, much of the underlying dynamics of synchrony as applied to ecology are not yet known. It is mathematically predicted that populations may move closer to and farther away from synchrony in cycles, a phenomenon known as phase difference modulation, but this has not been tested in ecological models. Here, we test for phase difference modulation using a mathematical model of metapopulations, and evaluate its effects on stability. We find that intermediate values of dispersal and habitat heterogeneity produce phase difference modulation. Additionally, we show that it can occur in simulated metacommunities where all populations naturally fluctuate as well as in those with one that is naturally at equilibrium but fluctuates due to dispersal. Phase difference modulation was found to cause populations' amplitudes to vary, leading to cyclic patterns of local and regional variability. Our results highlight the importance of viewing synchrony as a dynamic phenomenon, with implications for how synchrony between populations is measured in the field.

The endoplasmic reticulum (ER) is an interconnected membrane network of tubules and sheets found in all eukaryotic cells. The formation of this interconnected network requires…
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The endoplasmic reticulum (ER) is an interconnected membrane network of tubules and sheets found in all eukaryotic cells. The formation of this interconnected network requires homotypic fusion of ER membranes, which is mediated by a family of dynamin-like large Atlastin GTPases, including mammalian Atlastins, yeast Sey1p and plant RHD3 (ROOT HAIR DEFECTIVE 3). As a plant member of Atlastin GTPases, the mechanistic basis of how RHD3 acts in ER membrane fusion process is largely unknown.In the first part of the thesis, a systemic structure-function analysis of the action of different RHD3 domains in mediating ER fusion was conducted. Similar to Atlastins in mammalian cells and Sey1p in yeast cells, RHD3 has a GTPase domain in the N-terminus, a 3-helix bundle (3HB) enriched middle domain, two transmembrane domains and amphipathic C-terminal domain. I found that the GTPase domain and the first and second 3HBs in the middle domain promote the dimerization of RHD3. This dimerization of RHD3 is required for efficient ER membrane fusion. On the other hand, the third and fourth 3HBs are required for the protein stability of RHD3. I also found that the transmembrane domains (TMs) of RHD3 not only serve as an ER membrane anchor, but also facilitate the oligomerization of RHD3. In the C-terminal tail of RHD3, there is an amphipathic helix. I revealed that this amphipathic helix has a special membrane anchoring ability and is required for efficient ER membrane fusion. With this work, I contribute to a deeper understanding of the mechanistic basis of action of RHD3 in mediated ER membrane fusion.To maintain the ER homeostasis, the fusion action of Atlastin GTPases must be regulated and balanced. However, the knowledge of the regulation of action of Atlastin GTPses is very limited. Lunapark proteins (LNPs), a family of proteins known to be required for maintaining a normal tubular ER morphology, have been recently found to act antagonistically with Sey1p in yeast cells. But the exact molecular mechanisms how LNPs antagonize the action of Atlastin GTPases remain unclear. In the second part of the thesis, I found that there are two LNP homologs, LNP1 and LNP2 in Arabidopsis, both of them interact with RHD3 on 3-way junctions of the ER. Loss of LNPs in Arabidopsis cause plants to grow short root hairs and to be dwarfed. Additionally, massive sheet ER containing dense 3-way junctions is found in the cells. LNP1 and LNP2 are localized to 3-way-junctions of the ER where they stabilize the newly formed 3-way junctions of the ER. I revealed that LNPs are recruited by RHD3, after the ER membrane fusion, to the newly formed 3-way junctions. They can inhibit the fusion action of RHD3 likely by promoting the protein degradation of RHD3. With this work, I propose a novel post-membrane fusion regulation for RHD3 in which the RHD3 protein is degraded by the action of LNPs to avoid excessive fusion for the generation of an interconnected ER with an open polygonal network.

▼ Intrinsically disordered proteins (IDPs) or Intrinsically disordered regions (IDRs) in proteins can exhibit a partially folded or unfolded state under physiological conditions but confer several functional advantages. IDRs can fold into a stable tertiary structure when bound to their partner molecule, a transition that can be promoted by post-translational modifications (PTMs). Intrinsic disorder is found in all domains of life but is prevalent in eukaryotes. This thesis investigates the composition and evolutionary behaviour of disordered regions that undergo disorder-to-order transition and the evolutionary trend of PTMs in IDRs across eukaryotes using computational methods and tools. Bioinformatical parsing of human folding-on-binding (FB) proteins into four subsets (ordered, FBs, disordered regions that surround FBs, and other disordered regions) was performed to examine whether the composition and evolutionary behaviour (across vertebrate orthologs) are different in these four subsets. This analysis revealed that compositionally, ordered protein regions are distinct from the three other subsets, but the FB regions are of comparable evolutionary conservation to the ordered regions. Disordered regions surrounding FB regions are more negatively charged and less conserved than their adjacent FB regions. The presented results suggest the role of hydrophilic or charged residues around FBs in steering FB regions towards the binding sites of partner molecules. The insights gained from analysis of evolutionary conservation for FBs provided motivation to examine a related question, namely the evolutionary conservation of PTMs in IDPs/IDRs, in comparison to PTMs in ordered regions. In another bioinformatical approach, the conservation and emergence of methylation, acetylation and ubiquitination sites in ordered and disordered regions were examined across 11 evolutionary clades down from the whole eukaryotic domain to the ape superfamily. These sites occur mainly at arginine and lysine residues. It was discovered that MAU PTM is a major driver of conservation for arginines and lysines in both ordered and disordered regions, across the 11 levels, most significantly across the mammalian clade. Furthermore, the emergence of a significant number of new lysine MAU sites is found in the disordered regions of proteins in deuterostomes and mammals. In histones, MAU sites exhibit a distinct significant conservation pattern evident as far back as the last common ancestor of mammals. In a separate multiple evolutionary level analysis of the experimentally-verified human FB regions, a significant enrichment of conserved ubiquitination sites in FB regions was identified at all evolutionary levels back as far as mammals. Similarly, FB regions showed a significant preference for sites with multiple MAU modifications when treated both as a sample of ordered and of disordered regions. These results indicate the need to consider sequence analysis of IDRs at multiple evolutionary levels in order to understand their complex evolutionary…
Advisors/Committee Members: Paul Harrison (Supervisor).

Parasites are a global issue across the globe. In the Canadian livestock industry, resistance to commonly used drugs leads to significant losses in productivity and…
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Parasites are a global issue across the globe. In the Canadian livestock industry, resistance to commonly used drugs leads to significant losses in productivity and profit. The study of anthelminthics such as ivermectin is thus beneficial to finding new ways to protect mammals from these parasites and to increase farming income. We are interested in genes that confer both ivermectin resistance and a dye-filling defective phenotype, called dyf genes, when mutated. Dyf genes are of interest because they have been shown to be associated with ivermectin resistance in the model nematode Caenorhabditis elegans and in parasites such as Haemunchus contortus, however the mechanisms of resistance remain unclear. In this project, we investigate three mechanisms by which the dyf genes might confer ivermectin resistance: either by altering drug entry, communication within the nervous system, and/or stress response pathways. Our results suggest that drug entry plays an important role in ivermectin resistance, although it is unclear whether importance lies in dye-filling neurons or neurons with direct connections to glutamate-gated chloride channels. Additionally, the subset of P-glycoprotein efflux pump genes pgp-1, pgp-2, pgp-3, pgp-9, and mrp-1 is essential in maintaining ivermectin resistance in the dyf-7 mutant.

► Epigenetic mechanisms, especially DNA methylation, have been typically studied in the domains of development and disease; however, recent studies have suggested that epigenetics may also…
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▼ Epigenetic mechanisms, especially DNA methylation, have been typically studied in the domains of development and disease; however, recent studies have suggested that epigenetics may also play important roles in ecological and evolutionary processes. In this thesis, I examine the roles of DNA methylation in facilitating animals responding to environmental change. I begin my thesis with a chapter that provides a review of empirical and theoretical studies analysing the effects of epigenetics on phenotypic plasticity and evolution in animals. This forms the background knowledge for the dissertation, and also helps to reveal knowledge gaps to be filled by my other chapters. I found that epigenetic patterns can be shaped by the environment both within and across generations, and that epigenetic variation can play a role in local adaptation. I also evaluate the evolutionary potential of epigenetic variation depending on its autonomy from genetic variation, and its transgenerational stability. During my literature review, I found that environmental effects on epigenetic variation have typically been assessed under laboratory conditions. Thus, to add to the limited but growing body of literature on epigenetics in natural populations of animals, I evaluate epigenetic responses to environmental conditions in three distinct empirical systems and ecological scenarios. In my second chapter, I investigate changes in genome-wide DNA methylation patterns of Trinidadian guppies (Poecilia reticulata) during the course of infection by the monogenean ectoparasite, Gyrodactylus turnbulli. I found an epigenetic signature of infection by ectoparasites, and identified unique methylation responses at distinct phases of infection. In my third chapter, I explore genome-wide DNA methylation variation of Anolis lizards (Anolis sagrei) during the early stage of colonization of new habitats. I found that the magnitude of epigenetic variation was dependent on the environmental shift between new and source habitats, and discovered a potential relationship between epigenetic variation and physiological changes in populations at the earliest stages of colonization of new environments. Together with previous work, results from the two chapters suggest that patterns of DNA methylation can rapidly respond to environmental change, and that these methylation changes are involved in the regulation of critical genes and pathways. Although these findings highlight the importance of environmentally-mediated methylation changes, most genomic methylation patterns are static across tissues and throughout life, and some are even stable across generations. Thus, in my last chapter, I use threespine stickleback (Gasterosteus aculeatus) to characterise the distribution and function of constitutive methylation, and assess the amount of heritable methylation. I found a clear pattern of epigenetic variation across generations that is likely to be shaped by genetic variation. In addition, I found that constitutive methylation mapped to genes with functions relevant to…
Advisors/Committee Members: Rowan Barrett (Supervisor).

Accurate chromosome segregation requires both the correct assembly of the mitotic spindle, as well as the proper placement of the spindle relative to the plane…
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Accurate chromosome segregation requires both the correct assembly of the mitotic spindle, as well as the proper placement of the spindle relative to the plane of cell division. Interruption of either of these processes can lead to severe consequences such as cell death or, in high eukaryotes, cancer. Spindle assembly and spindle placement are largely driven by the function of microtubules. Microtubules are primarily nucleated from microtubule organizing centers (MTOCs) by an evolutionarily conserved protein complex: the gamma-tubulin ring complex (gamma-TuRC). While canonically involved in microtubule nucleation, the gamma-TuRC is also involved in numerous nucleation-independent cellular processes. How the gamma-TuRC is regulated such that it participates in both nucleation-dependent and -independent cellular functions is unclear. Here, we use the budding yeast, Saccharomyces cerevisiae, to investigate the regulation of gamma-tubulin through phosphorylation. We systematically characterized the phosphorylation sites of gamma-tubulin which is associated with the spindle pole body (the yeast MTOC) by creating phospho-mutants at each site both independently and in tandem. We found that the G1-specific phosphorylation sites, T130 and T227, contribute to chromosome attachment in a manner independent of microtubule dynamics. Additionally, we identified an intramolecular relationship between sites S360 and Y445 which regulates spindle assembly. Lastly, we identified a novel gamma-tubulin allele, Y362E, which participates in spindle placement through the regulation of astral microtubule number. Collectively, this work furthers our understanding of gamma-tubulin regulation and its role in the processes of spindle assembly and spindle placement. Additionally, as the majority of the phosphorylation sites in yeast gamma-tubulin are well-conserved across species, this regulation is likely an evolutionarily conserved mechanism to control gamma-tubulin function.

The properties of developmental systems that limit or bias the production of phenotypic variation, called structural or developmental constraints, can influence evolutionary responses to natural…
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The properties of developmental systems that limit or bias the production of phenotypic variation, called structural or developmental constraints, can influence evolutionary responses to natural selection. However, evidence to support a significant role for such constraints in shaping phenotypic evolution on a macroevolutionary scale remains elusive. The study of morphological variation is paramount to addressing this question, since morphology provides much of the primary information for evolution across geological time. This thesis presents four case-studies examining constraints on morphological evolution using morphometric analyses. Chapter 1 tests whether the magnitude and pattern of morphological integration and modularity was a limiting or facilitating factor during the diversification of pectoral fin morphology in teleost fishes. I find that higher integration is positively associated with shape disparity and evolutionary rate, supporting a facilitating effect of integration. Chapter 2 investigates the capacity of developmental plasticity to alter allometric scaling of the skeleton in an amphibious fish. I find that fish reared in a terrestrial environment show changes in the size and shape of their pectoral girdle bones compared to aquatically-reared fish. Chapter 3 proposes a simple method of incorporating a priori hypotheses of geometric constraints into a model of continuous trait evolution and compares this model to observed cranial morphologies in theropod dinosaurs. The model reveals a trend of convergent evolution in hypercarnivorous taxa and also highlights the novel skull morphologies of some lineages. Chapter 4 ends the thesis with a critical evaluation of principal component analysis as a tool for visualising multivariate morphological variation. I find that non-linear dimensionality reduction techniques often outperform principal component analysis, but that caution should always be used then interpreting simplified visualisations of high-dimensional data. Using the analytical toolbox of morphometrics, this thesis demonstrates that the investigation of constraints and structural aspects of morphology contribute unique insights on the origin of macroevolutionary patterns.

Nature is a wealth of inspiration for biomimetic and actuating devices. These devices are, and have been, useful for advancing society, such as giving humans…
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Nature is a wealth of inspiration for biomimetic and actuating devices. These devices are, and have been, useful for advancing society, such as giving humans the capability of flight, or providing household products such as velcro. Among the many biomimetic models studied, plants are interesting because of the scope of functions and structures produced from combinations of the same basic cell wall building blocks. Hierarchical investigation of structure and composition at various length-scales has revealed unique micro and nano-scale properties leading to complex functions in plants. A better understanding of such micro and nano-scale properties will lead to the design of more complex actuating devices, including those capable of multiple functions, or those with improved functional lifespan.In this thesis, the resurrection plant Selaginella lepidophylla is explored as a new model for studying actuation. S. lepidophylla reversibly deforms at the organ, tissue, and cell wall level as a physiological response to water loss or gain, and can repeatedly deform over multiple cycles of wetting and drying. Thus, it is an excellent model for studying properties leading to reversible, hierarchical (i.e., multi length-scale) actuation. S. lepidophylla has two stem types, inner (developing) and outer (mature), that display different modes of deformation; inner stems curl into a spiral shape while outer stems curl into an arc shape.In depth investigation of S. lepidophylla revealed that morphological and compositional gradients result in hierarchical stiffness gradients leading to differential tissue swelling/shrinking and, ultimately, directional stem (un)curling. Morphological 2gradients are observed at the tissue level between adaxial and abaxial stem sides, and include changes in tissue density, cell shape, size, and cell angle relative to the stem axis. Compositional gradients are observed along the length of the stem from tip to base at both tissue and cell wall levels and include changes in lignification and xylan distribution. Comparison between inner and outer S. lepidophylla stem types showed that morphological gradients are most likely involved in directional stem bending while compositional gradients appear to contribute to the degree of stem curling.

► Living in groups has many advantages for individuals, such as access to social information or protection against predators. The function and evolution of sociality and…
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▼ Living in groups has many advantages for individuals, such as access to social information or protection against predators. The function and evolution of sociality and social information use have received abundant attention in the past years. However, the neural mechanisms underlying these processes have not been as widely examined across taxa, which is crucial to understand their evolution in vertebrates. In this thesis, I explored the neuroendocrinal mechanisms of grouping and the neural mechanisms of social information use in the Trinidadian guppy (Poecilia reticulata), a species with extremely well-studied evolutionary ecology. In Chapter 2, I compared immediate early-gene (IEG) expression across brain areas of the social decision-making network when guppies were exposed to a large or small group of conspecifics, or isolated. I found higher IEG expression in the preoptic area when guppies were exposed to the large group, compared to control (isolated guppies). The preoptic area regulates social behaviour in vertebrates and is also the only region in the teleost brain producing the nonapeptides isotocin and vasotocin. Since nonapeptide homologues modulate grouping behaviour in birds and are neuromodulators of mammalian social behaviour, I hypothesized vasotocin and isotocin would have effects on grouping in fish. Thus, in Chapter 3, I developed a technique to centrally administer nonapeptides in the brain of guppies and investigated their effects on grouping. I found opposing effects of the two nonapeptides, with isotocin increasing and vasotocin reducing grouping, at 90 minutes after administration, consistent with these neuromodulators playing a role in a fundamental social behaviour in fish. An advantage of grouping behaviour is that it may facilitate the transmission of information between conspecifics. Thus, in Chapter 4, I explored the neural mechanisms of social information use by studying the brain areas activated when 'demonstrator' guppies were exposed directly to alarm substance (i.e., a reliable social cue released from damaged skin) and when they observed a conspecific reacting to alarm substance. I found that alarm substance provoked typical antipredator behaviour such as freezing and area avoidance, while visual exposure to alarmed conspecifics induced a preference to use the same area as the alarmed conspecifics. I examined patterns of IEG expression across six areas of the brain, finding that the demonstrators had higher overall expression of egr-1 compared to observers and control fish, and different patterns of correlated expression in demonstrators and observers, suggesting that coordinated activation across regions is involved in processing and modulating responses to social alarm cues in guppies. In summary, my thesis 1) establishes a basis for the study of the neural mechanisms of grouping and social information use in teleosts by highlighting the brain areas activated during these processes, 2) provides evidence of the conserved effects of nonapeptides on grouping in a vertebrate lineage…
Advisors/Committee Members: Simon Reader (Supervisor).